The gonadotropin-releasing hormone receptor (GnRH-R), a member of the G- protein coupled receptor (GPCR) superfamily, is a key mediator of the reproductive neuroendocrine system. GnRH analogs have wide therapeutic applications, ranging from the treatment of cancer to infertility. Elucidating the structural domains of the receptor involved in ligand binding would facilitate the directed design of improved analogs and expand our understanding of other GPCRs as well. We have recently determined the primary sequence of the mouse GnRH-R through molecular cloning. We now propose to isolate the human GnRH-R and to integrate evolutionary, mutagenesis, computational molecular modeling the simulation studies to develop a working model of this receptor and its mechanism of ligand binding. In order to compare conserved structural motifs across mammalian species, partial length clones of the sheep and dog GnRH-Rs will be isolated. These "natural mutants" may provide important clues about structure and ligand binding by revealing which GnRH-R residues are conserved throughout mammalian evolution. Human GnRH-R mutagenesis studies will be undertaken using a combination of several conceptual approaches to guide the studies, including: (1) alteration of residues conserved throughout mammalian evolution, (2) alteration of residues in positions known to be critical for ligand binding of other classical and peptide GPCRs, (3) alteration of residues postulated to interact with GnRH based on the pharmacology of GnRH analogs and known receptor biochemistry and (4) domain alteration to test an evolving three-dimensional molecular model. Because G-protein interaction with intracellular domains of the receptor reciprocally affect ligand binding, both aspects will be studied in transfected mammalian cells. An antibody to the receptor will be produced for quantifying receptor expression and for studying receptor glycosylation. The molecular biological experiments will be performed in conjunction with the computational molecular modeling. This project will thus generate a working model of the transmembrane helix bundle of the receptor, will suggest the specific sites of interaction with GnRH agonists and antagonists and will set the stage for a fuller understanding of the mechanisms of receptor activation and signal transduction.